Introduction

Polyunsaturated fatty acids (PUFAs) play important roles not only as structural components of membrane phospholipids but also as precursors of the eicosanoids of signaling molecules, including prostaglandins, thromboxanes, and leukotrienes, which are essential for all mammals. Fish oils, animal tissues, and algal cells are conventional, relatively rich sources of C20 PUFAs, which are not present in plants. For practical purposes, however, these conventional sources are not satisfactory with regard to either the lipid contents or the PUFA contents of the resultant lipids. To find more suitable sources of PUFAs in microbes, the first attempts at PUFA production with γ-linolenic acid (GLA, 18:3n-6) as the target were performed in the UK (Ratledge, 1992) and Japan (Suzuki et al., 1981), with Mucor fungi being used. Since then, various PUFAs have been studied with the aim of effective production. For example, arachidonic acid (AA, 20:4n-6), dihomo-γ-linolenic acid (DGLA, 20:3n-6), and Mead acid (MA, 20:3n-9) are now commercially produced by using Mortierella fungi (Certik et al., 1998;Certik & Shimizu, 1999; Sakuradani et al., 2005b; Shimizu & Yamada, 1990; Yamada et al., 1992), and docosahexaenoic acid (22:6n-3), docosapentaenoic acid (22:5n-6), and eicosapentaenoic acid (EPA, 20:5n-3) by using marine microorganisms, Labyrinthulae, and microalgae (Certik & Shimizu, 1999; Kyle et al., 1992; Nakahara et al., 1996; Raghukumar, 2008; Ratledge, 2004; Singh & Ward, 1997; Spolaore et al., 2006; Yazawa et al., 1992). Although success in this area over the last 25 years has generated much interest in the development of microbial fermentation processes, manipulation of the lipid compositions of microorganisms requires new biotechnological strategies to obtain high yields of the desired PUFAs.

The genus Mortierella has been shown to be one of the promising single cell oil (SCO) sources rich in various types of C20 PUFAs (Amano et al., 1992; Shimizu & Jareonkitmongkol, 1995), after several Mortierella strains were reported to be potential producers of AA in 1987 (Totani & Oba, 1987; Yamada et al., 1987). In particular, several Mortierella alpina strains have been extensively studied for the practical production of AA (Shinmen et al., 1989). Some of them are now used for the commercial production of SCO rich in AA. Among them, M. alpina 1S-4 has a unique ability to synthesize a wide range of fatty acids and has several advantages not only as an industrial strain but also as a model for lipogenesis studies. The biosynthetic pathways for n-9, n-6, and n-3 PUFAs in M. alpina 1S-4 are shown in Fig. 2.1a. The main product of the strain, AA, is synthesized through the n-6 pathway, which involves Δ12 and Δ6 desaturases, elongase (EL2), and Δ5 desaturase. Depending on the conditions, the total amount of AA varies between 3 and 20 g/L (30–70% of the total cellular fatty acids), with 70–90% of the AA produced being present as triacylglycerols (Higashiyama et al., 1998, 2002; Shimizu et al., 2003b).

Here, we describe recent progress in the breeding of commercially important arachidonic acid-producing M. alpina strains, particularly approaches for creating desaturase and elongase mutants with unique pathways for PUFA biosynthesis involving conventional chemical mutagenesis and modern molecular genetics. Such mutants are useful not only for the regulation and overproduction of valuable PUFAs but also as excellent models for the elucidation of fungal lipogenesis.

Derivation of Mutants from M. alpina 1S-4

A wide variety of mutants defective in desaturases (Δ9, Δ12, Δ6, Δ5, and n-3) or elongase (EL1) or mutants with enhanced desaturase activities (Δ6 and Δ5) have been derived from M. alpina 1S-4 by treating the parental spores with N-methyl-N’-nitro-N-nitrosoguanidine (Jareonkitmongkol et al., 1992c). In addition, diacylglycerol-accumulating mutants and several lipid-excretive ones have been isolated by the same method. They are valuable both as producers of useful PUFAs (novel or already existing) and for providing valuable information on PUFA biosynthesis in this fungus (Certik et al., 1998). The main features of these mutants grown on glucose and the biosynthesis of various types of PUFAs by them are outlined below (see also Fig. 2.2).